CN104372009A - New pathogenic gene of Melkersson-Rosenthal syndrome, and coding protein and application thereof - Google Patents

Abstract

The invention relates to a new pathogenic gene-FATP1 gene related to Melkersson-Rosenthal syndrome. Particularly, the inventor uses a family suffered from Melkersson-Rosenthal syndrome as the study object, performs exon set sequencing and comparison on sick individuals and non-sick individuals in the family and detects a missense mutation (c.68C>G,p.Pro23Arg) in the FATP1 gene, which can cause changes of the FATP1 protein. On such basis, the invention provides a mutant FATP1 gene, a coding protein and application thereof, a vector comprising the mutant FATP1 gene, a host cell and a kit. The mutant FATP1 gene can be used for carrying out molecular diagnosis and sickening risk evaluation on Melkersson-Rosenthal syndrome. The mutant gene and coding protein thereof can also be used as a pharmaceutical target spot for treating Melkersson-Rosenthal syndrome.

Description

The Disease-causing gene that Melkersson Rosenthal syndrome is new and coded protein thereof and application

Technical field

The present invention relates to a kind of human variation's gene, particularly relate to the Disease-causing gene-FATP1 gene that a kind of Melkersson Rosenthal syndrome is new.The invention still further relates to the FATP1 gene of sudden change and coded protein thereof and application, comprise the carrier of the FATP1 gene of sudden change, host cell and test kit.

Background technology

Melkersson Rosenthal syndrome (Melkersson-Rosenthal Syndrome, MRS) (OMIM 155900) is the heredopathia of familial, (can be see: Meisel-Stosiek M in autosomal dominant inheritance feature, Hornstein OP, Stosiek N.Family study onMelkersson-Rosenthal syndrome.Some hereditary aspects of the disease andreview of literature.Acta dermato-venereologica.1990; 70 (3): 221-6.PubMedPMID:1972835.).This sick distinctive clinical manifestation comprises cheilitis granulomatosa, facioplegia and fissured tongue, (can be see: Elias MK also known as macrochilia-facial paralysis-fissured tongue syndrome, Mateen FJ, Weiler CR.The Melkersson-Rosenthal syndrome:a retrospective study ofbiopsied cases.Journal of neurology.2013Jan; 260 (1): 138-43.PubMed PMID:22836908; Talabi OA.Melkerssons-Rosenthal syndrome:a case report andreview of the literature.Nigerian journal of clinical practice.2011Oct-Dec; 14 (4): 477-8.PubMed PMID:22248954.).First this disease is described in nineteen twenty-eight by Melkersson, and he only noticed recurrent lip oedema and facial paralysis at that time.Until nineteen thirty Rosenthal using fissured tongue also as the symptom of this disease, just make this disease be familiar with completely.Kunstadter reports the case of 1 example half morbidity in 5 years old in nineteen sixty-five, single side face neural paralysis and facial edema is there is during its grandmother 68 years old, occur single side face neural paralysis and suspicious facial edema during its one's mother's sister 10 years old, finally recovering completely (can be see: Kunstadter RH.Melkersson's syndrome.A case report ofmultiple recurrences of Bell's palsy and episodic facial edema.American journalof diseases of children.1965Nov; 110 (5): 559-61.PubMed PMID:5846695.).Did Carr etc. report at least 4 have two generations to get involved and 1 MRS family having three generations to get involved (can be see: Carr RD.Is the Melkersson-Rosenthal syndrome hereditary? Archives ofdermatology.1966Apr; 93 (4): 426-7.PubMed PMID:5862634.).Lygidakis etc. report 1 family four generation 7 people for 1979 and get involved, wherein 1 example is father-son heredity, 1 example is that atavism (can be see: Lygidakis C, Tsakanikas C, Ilias A, Vassilopoulos D.Melkersson-Rosenthal's syndrome in four generations.Clinical genetics.1979Feb; 15 (2): 189-92.PubMed PMID:761419.).Smeets etc. report one 26 years old women for 1994 MRS typical clinical manifestations, and chromosome karyotype analysis has t (9; 21) (p11; P11) make a variation, its mother's karyotype is normal (can be see: Smeets E, Fryns JP, Van den Berghe H.Melkersson-Rosenthal syndrome and de novo autosomal t (9; 21) (p11; P11) translocation.Clinical genetics.1994 Jun; 45 (6): 323-4.PubMed PMID:7923865.).In recent years also there are several MRS family and case report (can be see: Liu R, Yu S.Melkersson-Rosenthal syndrome:a review of seven patients.Journal of clinicalneuroscience:official journal of the Neurosurgical Society of Australasia.2013Jul; 20 (7): 993-5.PubMed PMID:23664134.).

At present about the cause of disease of MRS and pathogenesis completely not clear, may with heredity, infects, the multiple factors such as vasomotoricity disorder that immunologic function disorder, anaphylaxis and autonomic nervous system regulate is relevant.

This disease all has report all over the world, and both sexes sickness rate is roughly equal, occurs symptom when children or youth.Macrochilia scope and varying degree, what have only has slight plumpness, 1 to the several times of reached at the healthy tissues had.Lip swelling can be paroxysmal, disappears very soon or recurrent exerbation, and continue a few hours or several weeks, antihistaminic is invalid.The recurrent hemiprosoplegia of MRS is one-sided or bilateral, is partial or thoroughness facial paralysis.30% ~ 80%MRS patient has fissured tongue, and tongue rear face has dark and crisscross ditch gap.The typical pathologic of this disease shows as lymphedema, non-cheesy epithelial cell granuloma.The histopathological examination display lymphocytic infiltration of upper lip biopsy and submucosa showed edema, there is obvious lymphocytic infiltration (can be see: Elias MK around in blood vessel and Lymphoid tissue, Mateen FJ, Weiler CR.TheMelkersson-Rosenthal syndrome:a retrospective study of biopsied cases.Journal of neurology.2013Jan; 260 (1): 138-43.PubMed PMID:22836908.).

MRS there is no effective treatment way, easily recurs.Although can there is spontaneous resolution of symptoms, complete remission rate is low.Upper flaps can cause permanent plumpness due to recurrent exerbation.Facial paralysis is peripheral, repeatedly occurs, how can recover completely, but prolonged and repeated outbreak can leave over masseter and frontalis atrophy and intractable facial paralysis etc.Local glucocorticoid skin can be adopted to damage interior injection for lip obvious tumefaction person, have certain curative effect but easily recur.Oral dapsone, Thalidomide or trypterygine have certain curative effect, and oral glucocorticoid effectively but general not exemplary application.Lip swelling obviously can be disappeared person completely, glucocorticosteroid (Betamethasone Valerate etc.) local injection can be tried out, usually can obtain partial rcsponse (can be see: Oudrhiri L, Chiheb S, Marnissi F, Zamiati S, Benchikhi H.Successful treatmentof Miescher's cheilitis in Melkersson-Rosenthal syndrome with betamethasoneinjections and doxycycline.The Pan African medical journal.2012; 13:75.PubMed PMID:23397029.Pubmed Central PMCID:3567407.).The reported 8 cases such as Dai C have the prosopoplegic MRS patient of recurrent, adopt through mastoid process facial nerve part lysis and average observation 3.3 years, wherein there are 7 routine facioplegia hierarchical restorations to I grade or II grade, (can be see: Dai C without recurrence, Li J, Yang S, Zhao L, Feng S, Li Y, et al.Subtotal facial nervedecompression for recurrent facial palsy in Melkersson Rosenthal syndrome.Acta oto-laryngologica.2014Apr; 134 (4): 425-8.PubMed PMID:24512460.).The report such as Stein J adopts A Damu monoclonal antibody (tumor necrosis factor alpha blocker) to treat MRS patient effectively (can be see: Stein J, Paulke A, Schacher B, Noehte M.An extraordinary form ofthe Melkersson-Rosenthal syndrome successfully treated with the tumournecrosis factor-alpha blocker adalimumab.BMJ case reports.2014; 2014.PubMed PMID:24827666.).To sum up, the Diagnosis and Treat correct to MRS is the huge challenge that clinician faces, and needs the joint efforts of multidisciplinary clinicist.Await further work about its definite cause of disease and pathogenetic seeking, the cause of disease and the Forming Mechanism that inherently disclose MRS are the keys finding MRS to treat.

Full-length genome exon order-checking (target exon group is caught) is a kind of sequence measurement of economy, and it mainly checks order to the coding region of human genome thus detects the rare new gene relevant with common disease.Because only check order 1% of full gene group, so both economical and (can be see: Choi M with higher covering efficiency and the degree of depth, Scholl UI, Ji W, Liu T, Tikhonova IR, Zumbo P, etal.Genetic diagnosis by whole exome capture and massively parallel DNAsequencing.Proc Natl Acad Sci U S A.2009Nov 10; 106 (45): 19096-101.PubMed PMID:19861545; Cooper GM, Goode DL, Ng SB, Sidow A, BamshadMJ, Shendure J, et al.Single-nucleotide evolutionary constraint scores highlightdisease-causing mutations.Nat Methods.2010Apr; 7 (4): 250-1.PubMed PMID:20354513.Pubmed Central PMCID:3145250.Epub 2010/04/01.eng.).The function variation of most of single-gene disorders known at present betides the exon of Disease-causing gene (can be see: Stenson PD, Ball EV, Mort M, Phillips AD, Shiel JA, Thomas NS, et al.HumanGene Mutation Database (HGMD): 2003update.Hum Mutat.2003Jun; 21 (6): 577-81.PubMed PMID:12754702.Epub 2003/05/20.eng; Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, et al.Targetedcapture and massively parallel sequencing of 12human exomes.Nature.2009Sep 10; 461 (7261): 272-6.PubMed PMID:19684571.), therefore the order-checking of full-length genome exon is considered to the important technology making up positional cloning.Since 2009, exon trapping system and sequencing system has successfully been utilized to find or demonstrate some single-gene disorders (Freeman – Sheldon syndromes both at home and abroad, Miller syndrome, Kabuki syndrome, BVVL syndrome, reverse property acne, spinocerebellar ataxia, Schinzel-Giedion syndrome, non-syndrome is deaf) Disease-causing gene (can be see: Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, et al.Exome sequencing identifies the cause of amendelian disorder.Nat Genet.2009Jan, 42 (1): 30-5.PubMed PMID:19915526, Ng SB, Bigham AW, Buckingham KJ, Hannibal MC, McMillin MJ, Gildersleeve HI, et al.Exome sequencing identifies MLL2mutations as a causeof Kabuki syndrome.Nat Genet.2010Sep, 42 (9): 790-3.PubMed PMID:20711175.Epub 2010/08/17.eng, Johnson JO, Gibbs JR, Van Maldergem L, Houlden H, Singleton AB.Exome sequencing in brown-vialetto-van laeresyndrome.Am J Hum Genet.2010Oct 8, 87 (4): 567-9.PubMed PMID:20920669.Epub 2010/10/06.eng, Liu Y, Gao M, Lv YM, Yang X, Ren YQ, Jiang T, et al.Confirmation by Exome Sequencing of the Pathogenic Role ofNCSTN Mutations in Acne Inversa (Hidradenitis Suppurativa) .J InvestDermatol.2011Mar 24, 131 (7): 1570-2.PubMed PMID:21430701.Epub2011/03/25.Eng, Wang JL, Yang X, Xia K, Hu ZM, Weng L, Jin X, et al.TGM6identified as a novel causative gene of spinocerebellar ataxias usingexome sequencing.Brain.2010Dec, 133 (Pt 12): 3510-8.PubMed PMID:21106500.Hoischen A, van Bon BW, Gilissen C, Arts P, van Lier B, Steehouwer M, et al.De novo mutations of SETBP1cause Schinzel-Giedionsyndrome.Nat Genet.2010May 2.PubMed PMID:20436468, Walsh.WholeExome Sequencing and Homozygosity Mapping Identify Mutation in the CellPolarity Protein GPSM2as the Cause of Nonsyndromic Hearing Loss DFNB82.Ame J Hum Gene.2010.).Wherein have some be to combine with the order-checking of full-length genome exon the Disease-causing gene finding monogenic disease with Linkage mapping method (can be see: Alvarado DM, Buchan JG, Gurnett CA, Dobbs MB.Exome sequencing identifies an MYH3mutation in afamily with distal arthrogryposis type 1.J Bone Joint Surg Am.2011Jun1; 93 (11): 1045-50.PubMed PMID:21531865.Epub 2011/05/03.eng; ZuchnerS, Dallman J, Wen R, Beecham G, Naj A, Farooq A, et al.Whole-ExomeSequencing Links a Variant in DHDDS to Retinitis Pigmentosa.Am J HumGenet.2011Feb 2; 88 (2): 201-6.PubMed PMID:21295283.Epub 2011/02/08.Eng; Otto EA, Hurd TW, Airik R, Chaki M, Zhou W, Stoetzel C, et al.Candidate exome capture identifies mutation of SDCCAG8as the cause of aretinal-renal ciliopathy.Nat Genet.2010Sep 12; 42 (10): 840-50.PubMed PMID:20835237; Ostergaard P, Simpson MA, Brice G, Mansour S, Connell FC, Onoufriadis A, et al.Rapid identification of mutations in GJC2in primarylymphoedema using whole exome sequencing combined with linkage analysiswith delineation of the phenotype.J Med Genet.2011Jan 25; 48 (4): 251-5.PubMed PMID:21266381.Epub 2011/01/27.Eng; Bolze A, Byun M, McDonald D, Morgan NV, Abhyankar A, Premkumar L, et al.Whole-Exome-Sequencing-Based Discovery of Human FADD Deficiency.Am JHum Genet.2010Dec 10; 87 (6): 873-81.PubMed PMID:21109225.).Full exon sequencing technologies screens the variation with disease-related by means of only checking order to the full exon of several individuality (comprising patient and normal control) seldom, and its success ratio greatly promotes.

Therefore, the Disease-causing gene of Melkersson Rosenthal syndrome (MRS) is found in this research and utilization exon group sequencing technologies district, to providing brand-new theoretical foundation for the patient treatment of MRS, enrich and improve the diagnostic process of MRS disease, thus provide more support and reference to the clinical diagnosis of MRS patient.

Summary of the invention

Main purpose of the present invention is the new Disease-causing gene differentiating Melkersson Rosenthal syndrome (MRS), orients the gene mutation site of this disease.On this basis, MRS Disease-causing gene and coded protein thereof and application are provided, comprise the carrier of MRS Disease-causing gene, host cell and test kit, to carry out molecular diagnosis and risk evaluation to Melkersson Rosenthal syndrome, there is provided the target spot of design medicine for treating this disease further, the mechanism of causing a disease simultaneously for illustrating this disease is provided fundamental basis.

Therefore, on the one hand, the invention provides the FATP1 gene of sudden change, the FATP1 gene order of described sudden change has at least 1 non-silent mutation compared with SEQ ID NO:1, and the FATP1 gene of described sudden change causes the generation of Melkersson Rosenthal syndrome; Described non-silent mutation be selected from insertion, disappearance and displacement in one or more.

In a preferred embodiment, described non-silent mutation is that the C of the 68th of SEQ ID NO:1 sports G, and its sequence is as shown in SEQ ID NO:2.This sudden change belongs to missense mutation, makes the proline(Pro) of the aminoacid sequence of FATP1 genes encoding the 23rd become arginine.

Second aspect, present invention also offers the protein of SEQ ID NO:2 sequence encoding, and this sequence changes from the 23rd, and described protein has 646 amino acid, and its aminoacid sequence is as shown in SEQ IDNO:3.

In a third aspect of the present invention, provide the carrier containing said mutation FATP1 gene.

In a fourth aspect of the present invention, provide by the host cell of above-mentioned vector or transduction or the host cell that directly transformed by the FATP1 gene of said mutation or transduce.

In a fifth aspect of the present invention, the FATP1 gene providing above-mentioned sudden change is as the drug target for the treatment of Melkersson Rosenthal syndrome or the application prepared in Melkersson Rosenthal syndrome kit for diagnosing diseases.

In a sixth aspect of the present invention, provide a kind of for diagnosing the test kit of Melkersson Rosenthal syndrome, described test kit comprises can the primer of specific amplification FATP1 gene, or can the probe of specific detection FATP1 transgenation.

In a preferred embodiment, described primer or probe sequence are the sequence that in the sequence shown in SEQ ID NO:1 or 2, the 68th front and back 15 ~ 30bp grows.

In a seventh aspect of the present invention, provide a kind of antibody, described antibody is combined with protein specific according to claim 3, and does not act on the protein of normal FATP1 coded by said gene.

In a eighth aspect of the present invention, provide a kind of Melkersson Rosenthal syndrome therapeutical agent, described therapeutical agent comprises antibody described above.

FATP1 transgenation can cause the morbidity of Melkersson Rosenthal syndrome by exon group sequencing technologies Late Cambrian in the present invention.On the one hand, whether carry FATP1 gene pathogenic mutation by detecting experimenter, can early screening Melkersson Rosenthal syndrome mutator gene carrier, provide prenatal and postnatal care to instruct; Also can be Melkersson Rosenthal syndrome patient and molecular diagnosis foundation is provided.Especially, diagnostic kit provided by the present invention can be used for predicting quickly and efficiently or diagnosing Melkersson Rosenthal syndrome.On the other hand, FATP1 gene is further investigate with the pathogenesis of clear and definite MRS, and explores effective treatment means, and fundamentally solution MRS provides brand-new path without the present situation of effective treatment means; The third aspect, the present invention can provide possible drug target for treatment Melkersson Rosenthal syndrome.

Accompanying drawing explanation

Fig. 1 is certain four generation autosomal dominant inheritance MRS pedigree chart, and wherein square represents the male sex, and circle represents women, and band oblique line is dead, and solid is diseased individuals, and hollow is normal individual.

Fig. 2 is the clinical manifestation of patient in MRS pedigree chart in Fig. 1, and wherein A, B are the clinical manifestation of III 3 patients, and C is the clinical manifestation of II 5, and D is the clinical manifestation of II 3.

Fig. 3 is MRS patient's lip histopathology performance (light microscopic HE × 200).

Fig. 4 is the sequencing result of some individuals in MRS pedigree chart in Fig. 1, and wherein, A is the sequencing result of patient II 3, and B is the sequencing result of patient II 5, and C is the sequencing result of patient III 3, and D is the sequencing result of normal individual II 4.

Embodiment

Below in conjunction with embodiment, the present invention is made a more detailed description; but below describe only for the explanation of making an explanation property of the present invention; do not carry out any restriction to protection scope of the present invention, the scope that protection scope of the present invention limits with claims is as the criterion.

In the present invention, unless otherwise stated, Science and Technology noun used herein has the implication that those skilled in the art understand usually.Further, molecular genetics used herein, nucleic acid chemistry and molecular biology relational language and laboratory operation step are widely used term and conventional steps in corresponding field.Meanwhile, in order to understand the present invention better, provide definition and the explanation of relational language below.

In the present invention, " FATP1 gene " is fatty acid transport protein 1 (fatty acid transportprotein 1) gene, the FATP1 gene of people is positioned on No. 19 karyomit(e)s of human body, 646 amino acid of encoding, and wherein conservative row district coding has 311 amino acid.The protein of FATP1 genes encoding is fatty acid transport protein 1, it is the transmembrane protein integrated, belong to fatty acid transport protein family (FATPs) member, participate in lipid acid transmembrane transport and fatty acid metabolism, it is one of key gene affecting lipid content, this albumen is mainly expressed in fatty tissue, is also found at heart and skeletal muscle.

In the present invention, term " exon " refers to the part be retained in ripe mRNA, and namely ripe mRNA corresponds to the part in gene.Intron is the part be sheared in the mRNA course of processing, does not exist in ripe mRNA.Exon and intron are all for gene, and the part of coding is exon, and what do not encode is intron, and intron does not have hereditary effect.

In the present invention, term " exon trapping " and " chip hybridization " are used interchangeably, and refer to the process that the DNA fragmentation of probe to Exon region, library carries out specificity selection and combination.DNA molecular is double-strand under normal circumstances, and before therefore catching, DNA molecular must become strand, is generally make its sex change by heating and reach object of unwinding, and the DNA molecular unwind is rapidly cooled, and namely keeps single-chain state.Carry out catching hybridization at hybridization platform and chip after the sex change of library.Molecular hybridization is carried out under strict conditions between DNA fragmentation containing exon region and the probe being fixed on chip.Preferably, on chip, the concentration of probe molecule will far away higher than concentration of target molecules.After waiting to hybridize, collected the sequence purifying of catching by methods such as sex change, obtain from the sequential mixture after catching.

In the present invention, " high-flux sequence " refers to that employing three kinds of s-generation order-checking platforms carry out high-flux sequence: the SOLID etc. of 454FLX (Roche company), Solexa Genome Analyzer (Illumina company) and Applied Biosystems company.The common feature of these platforms is high sequencing throughput, relative to 96 road kapillary order-checkings of tradition order-checking, high-flux sequence is once tested and can be read 40 ten thousand to 400 ten thousand sequences, according to the difference of platform, do not read length from 25bp to 450bp not etc., therefore different order-checking platforms, in once testing, can read the base number that 1G to 14G does not wait.

In the present invention, term " DNA library " refers to and interrupts genomic object fragment, obtains the DNA fragmentation mixture that a group has a certain size.The preparation method of DNA library is well known to those skilled in the art.In a preference, stopping pregnancy thing of can also fighting each other, end are repaired product, joint product and enriched product and are carried out purifying.Certain change is carried out to the condition of reaction or optimizes also within those skilled in the art's limit of power.

In the present invention, term " sudden change " refers to that the FATP1 polynucleotide sequence of wild-type changes, and becomes varient, and varient can be that natural generation or non-natural occur.Varient comprises and replaces varient, Deletion variants and insertion varient.In the present invention, a certain varient can be accomplished in several ways, such as, obtain the incomplete varient of wild-type gene encodes, can, by inserting base in wildtype gene sequence, make certain codon become terminator codon, or the mode of direct lack part sequence realize.In the present invention, term " non-silent mutation " refers to the transgenation except silent mutation, includes but not limited to missense mutation, nonsense mutation and phase shift mutation etc.

The invention still further relates to and described FATP1 nucleotide hybridization and have at least 80% between two sequences, preferably at least 90%, the more preferably polynucleotide of at least 95%, at least 99% homology.The present invention be more particularly directed to polynucleotide interfertile with polynucleotide of the present invention under strict conditions.

Wild-type of the present invention or saltant type FATP1 Nucleotide full length sequence or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.

In the present invention, " carrier " includes but not limited to cloning vector and expression vector.In a preferred embodiment, described carrier is such as plasmid, clay, phage, coemid etc.In a preferred embodiment, described carrier is obtained commercially.In a preferred embodiment, described carrier comprises and the expression control sequenc that is connected of the FATP1 genes being operational of said mutation ground, such as but not limited to promotor, and enhanser and terminator.In a preferred embodiment, described carrier optionally also comprises selective marker.

Comprise the carrier of above-mentioned suitable DNA sequence dna and suitably promotor or control sequence, may be used for transforming suitable host cell, with can marking protein.This type of host cell includes but not limited to, prokaryotic cell prokaryocyte is Bacillus coli cells such as, and eukaryotic cell such as yeast cell, insect cell, vegetable cell and zooblast (such as, as mammalian cell, mouse cell, people's cell etc.).Host cell of the present invention can also be clone.

The invention still further relates to FATP1 mutein, because in nucleotide sequence shown in SEQ ID NO:l, the C of the 68th sports G, the proline(Pro) of the aminoacid sequence of FATP1 genes encoding the 23rd is made to become arginine, FATP1 mutein of the present invention has the sequence shown in SEQ ID NO:3, and the displacement of this site amino acids makes the FATP1 protein after suddenling change not have the normal function of wild-type FATP1 protein.FATP1 mutein of the present invention can be recombinant protein, natural protein, synthetic protein, preferred recombinant protein, namely recombinant technology is used to produce from protokaryon or eucaryon host (such as, bacterium, yeast, higher plant, insect and mammalian cell).

In the present invention, FATP1 mutein also relate to have with FATP1 mutein identical function, the variant form of SEQ ID NO:3 sequence.Variant form comprises: homologous sequence, conservative variant, induced mutants etc.Invention also relates to FATP1 mutein analogue.These analogues and FATP1 mutain qualitative difference can be the difference on aminoacid sequence, also can be the difference do not affected on the modified forms of sequence, or have both at the same time.

As well known to those skilled in the art, Disease-causing gene tool is of use in many ways.Therefore, the purposes of sudden change FATP1 gene provided by the invention includes but not limited to: the drug target being used as treatment Melkersson Rosenthal syndrome; Prepare in the diagnostic kit of Melkersson Rosenthal syndrome; For generation of disease animal model; For treatment Melkersson Rosenthal syndrome provides new pathways of drug action.

" test kit " of the present invention refers to those skilled in the art with FATP1 wild-type or mutant nucleotide for gene probe, according to the principle of gene recombination, can detect in biological specimen and whether there is the nucleotide sequence with this probe sequence complementation, therefore use this test kit can detect in sample whether there is gene mutation site of the present invention.Test kit generally comprises: primer, probe, nucleic acid chip, specification sheets etc., also may comprise the damping fluid compatible with activeconstituents, carrier or medium etc.

In the present invention, " primer " refers to the polynucleotide passage for the target nucleic acids that increases in PCR reaction, it typically is oligonucleotide, such as containing at least 5 bases, the polynucleotide passage of such as 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50 or more base.Primer need not with goal gene to be amplified or its complementary strand complete complementary, as long as it can specific amplification goal gene.As used herein, term " specific amplification " refers to that primer can react amplifying target genes by PCR, and other genes that do not increase.Such as, specific amplification FATP1 gene refers to, in PCR reaction, primer only increases FATP1 gene, and other genes that do not increase, the design of this type of primer well known to a person skilled in the art.

In the present invention, term " probe of specific detection FATP1 transgenation " refers to that probe can be distinguished the FATP1 gene containing sudden change and not contain the FATP1 gene suddenlyd change.Generally speaking, can, by controlling the stringency of hybridization conditions, probe can be distinguished containing the gene suddenlyd change and not containing the gene suddenlyd change.Such as, under high stringency conditions, with the probe of FATP1 gene exact complementarity can with not containing the FATP1 gene recombination suddenlyd change, and not with the FATP1 gene recombination of sudden change even only comprising a point mutation, thus the two to be distinguished.Equally, the probe with the FATP1 gene exact complementarity suddenlyd change can also be designed, thus its under high stringency conditions with the FATP1 gene recombination of sudden change, and not with not containing the FATP1 gene recombination that suddenlys change.In biology field, design and the hybridization technique of probe are known.

The present invention relates to and there is specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody to the FATP1 protein of sudden change.Here, " specificity " refers to that antibody capable is incorporated into the FATP1 protein of sudden change.Preferably, refer to that those can be combined with the FATP1 protein of sudden change or fragment but nonrecognition and be incorporated into the antibody of FATP1 protein associated antigen molecule of wild-type.The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises and have immunocompetent antibody fragment, as Fab, or (Fab) 2 fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered Single Chain Fv Molecule A or chimeric antibody.Antibody of the present invention can be prepared by the various technology that those skilled in that art are known.

The FATP1 protein antibody of sudden change of the present invention can be used for identifying the FATP1 protein suddenlyd change.Such as, the FATP1 protein specific antibodies of sudden change can be marked with a kind of detectable molecule such as fluorescein isothiocyanate (FITC), then allow sudden change FATP1 protein specific antibodies and sample contacts, go out the sample be combined with the FATP1 protein specific antibodies of sudden change again with fluorescent microscope or flow cytomery, thus provide foundation and guidance for whether prediction or diagnosis patient exist idiopathic Melkersson Rosenthal syndrome.

The FATP1 protein antibody of sudden change of the present invention can also be used to the FATP1 protein of neutral mutation.If the suddenly change existence of FATP1 protein of the MRS of certain body and the present invention is relevant to have enough data to show, can consider to neutralize these pathogenic mutations FATP1 protein molecule with the FATP1 protein-specific antibody of sudden change, thus the illness of reduction of patient.

Embodiment 1: sample acquisition

Contriver finds and identifies the Melkersson Rosenthal syndrome family (as shown in Figure 1) of an autosomal dominant inheritance, totally 4 generation 20 people, and wherein 3 people are patient's (individual shown in solid black in Fig. 1).Propositus is 30 years old women (III 3), from 12 years old left side face occur twice facial paralysis, through treatment recover completely, after recurred again; There is unknown cause swelling in lip, repeatedly occur again especially easily occurring (as shown in Figure 2 A) when there being external stimulus etc. from before 10 years later; Namely tongue has staggered crack since the childhood, occasionally has pain (as shown in Figure 2 B); Lip histopathology has the granuloma sample of chronic non-caseous necrosis to change (as shown in Figure 3), and above-mentioned phenotype meets MRS.Through questioning closely family history, find that II 3 (as shown in Figure 2 D) and II 5 (as shown in Figure 2 C) all have fissured tongue to change.

Contriver is using the normal person of phenotype (II 7) in 2 patients (II 5 and III 3) and 1 family as research sample, and the peripheral blood gathering every research object is as study sample, adds EDTA anti-freezing ,-80 DEG C of preservations.All blood samples all sign genus Informed Consent Form, and obtain Ethics Committee's approval.

Embodiment 2: prepared by sample DNA

Gather the peripheral blood of the normal person of phenotype (II 7) in Melkersson Rosenthal syndrome patient (II 5 and III 3) and family thereof, adopt OMEGA Blood DNA Midi Kit whole blood DNA to extract test kit and extract DNA from peripheral blood sample, extraction step is as follows:

(1) get 2ml whole blood sample, add 150ul OB Protease, 2.1ml Buffer BL and 20ulRNase A, top speed whirlpool 1 minute, thoroughly mixes.

(2) 65 DEG C of water-bath 15-20 minute, and in water-bath process whirlpool 5 times.

(3) add 2.2ml dehydrated alcohol, top speed whirlpool 30 seconds, thoroughly mixes.

(4) 3.5ml lysate is moved into the 15ml centrifuge tube of band Filter column, 4000 leave the heart 5 minutes, take out Filter column, outwell filter liquide, put back to Filter column.

(5) the 3rd step residue lysate is added the 15ml centrifuge tube of band Filter column, 4000 leave the heart 5 minutes, take out Filter column, outwell filter liquide, put back to Filter column.

(6) add 3ml HB Buffer, washing and filtering post, 4000 leave the heart 5 minutes, take out Filter column, outwell filter liquide, put back to Filter column.

(7) add 3ml DNA Wash Buffer, 4000 leave the heart 5 minutes, take out Filter column, outwell filter liquide, put back to Filter column.

(8) again add 3ml DNA Wash Buffer, 4000 leave the heart 5 minutes, take out Filter column, outwell filter liquide, put back to Filter column.

(9) 4000 leave the heart 15 minutes, dry Filter column.

(10) Filter column is moved to new 15ml centrifuge tube, add the ElutionBuffer of 500ul 70 degrees Celsius, room temperature leaves standstill 5 minutes, and 4000 leave the heart 5 minutes, collects the filtered liquid containing DNA.

(11) again Filter column is moved to new 15ml centrifuge tube, add the Elution Buffer of 500ul 70 degrees Celsius, room temperature leaves standstill 5 minutes, and 4000 leave the heart 5 minutes, collects the filtered liquid containing DNA.

(12) utilize concentration and the purity of spectrophotometer measurement DNA, the OD260/OD280 of each sample genomic dna of gained is all between 1.7 ~ 2.0, and concentration is no less than 200ng/ul, and total amount is no less than 30 μ g.

Embodiment 3: exon trapping and order-checking

Contriver checks order in conjunction with the exon group sequence of Solexa high throughput sequencing technologies to the patient of the embodiment 1 chosen with NimbleGen SeqCap EZ Exome (44M), specific as follows:

1) genomic dna is broken at random the fragment of about 200-300bp, connects top connection subsequently at fragment two ends respectively and prepare Hybrid Library (the Illumina/Solexa standard provided see http://www.illumina.com/ builds storehouse specification sheets).

2) library purified after carry out hybridization enrichment through the linear amplification of ligation-mediated PCR (ligation-mediated PCR (LM-PCR)) and Biotinylated DNA Library, then after the linear amplification of LM-PCR, carry out upper machine order-checking.Order-checking platform is Illumina Hiseq 2000, reading length is 90bp, the average order-checking degree of depth of each sample reaches 100 × (namely in each sample, the data volume dropping on target area after comparison removes duplication with reference to genomic clean data is 100 times of the target area size that probe covers, and target area and size thereof depend on the mode of catching and building storehouse).

3) raw data obtained after order-checking is processed by Illumina basecalling Software 1.7, depollute through filtration, use SOAPaligner 2.20# comparison with reference to genome (can with reference to Li R, Li Y, KristiansenK, et al, SOAP:short oligonucleotide alignment program.Bioinformatics 2008,24 (5): 713-714; Li R, Yu C, Li Y, et al, SOAP2:animproved ultrafast tool for short read alignment.Bioinformatics 2009,25 (15): 1966-1967, by reference to mode it is incorporated in full herein), to obtain comparison to the unique aligned sequences on genome.Then utilize SOAP snp (can be see: Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively parallel whole-genomeresequencing.Genome Res 2009,19 (6): 1124-1132, by reference to mode it is incorporated in full herein) determine the genotype of target region.Indel (insertion-deletion, insertion/deletion mark) adopt BWA (by Burrows-Wheeler ratio of transformation to) (version 0.5.9-r16) comparison to reference genome Hg19 (snp132), then utilize GATK (Genome Analysis Toolkit) (versionv1.0.4705) to determine the type of indel.(can see Li H, Durbin R.Fast and accuratelong-read alignment with Burrows-Wheeler transform.Bioinformatics 2010; 26 (5): 589-595; McKenna, A, Hanna M, Banks E, et al.The genome analysistoolkit:a MapReduce framework for analyzing next-generation DNAsequencing data.Genome Research 2010; 20 (9): 1297-1303).

Subsequently four public database (dbSNP (v137): http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp137. txt.gz. are passed through to result; Thousand personal data storehouse: ftp: //ftp.1000genomes.ebi.ac.uk/vol1/ftp or ftp: //ftp-trace.ncbi.nih.gov//1000genomes/ftp; Hapmap:ftp: //ftp.ncbi.nlm.nih.gov/hapmap; YH database: the filtration of http://yh.genomics.org.cn, removes all known and gene frequency variations of being greater than 0.005 in a database.By the normal sample of comparison, remove all known variations, the variation of synonym variation and non-coding region, and utilize SIFT software to carry out SNP function prediction, finally obtain transgenation (FATP1c.68C>G, p.Pro23Arg that 3 may have pathogenic meaning; FLI1c.74C>T, p.Ala25Val; CACNA1Hc.4819C>T, p.Arg1607Cys).

Embodiment 4:Sanger method sequence verification

Because the order-checking of exon group exists false positive to a certain degree, therefore we verify above-mentioned 3 transgenations with pathogenic meaning further, and select the normal population checking in MRS family and outside MRS family at this, concrete steps are as follows:

1.DNA extracts

Gather the peripheral blood of the normal person of all phenotypes and 200 irrelevant normal individuals in the MRS patient (II 5 and III 3) in embodiment 1, its family respectively, genomic dna is extracted according to the method for embodiment 2, measure afterwards and extract DNA purity and concentration, the may be used for PCR of OD260/280 between 1.8-2.0 of DNA detects.

2. design of primers and PCR reaction

Design of primers reference men and women genoid data unit sequence storehouse hg19/build36.3, under being specifically shown in.

PCR detects the SapphireAmp Fast PCR Master Mix detection kit adopting TaKaRa company.PCR reaction system is as following table:

Reaction conditions:

Wherein, annealing temperature sets respectively according to often pair of primer, and the annealing temperature of FATP1 primer is set to 62.9 DEG C, and the annealing temperature of FLI1 primer is set to 57.6 DEG C, and the annealing temperature of CACNA1H primer is set to 61.7 DEG C.

3.DNA checks order

PCR reaction product is carried out 2% agarose gel electrophoresis; observe under ultraviolet lamp have clear bright band and stripe size meet target product size carry out Sanger order-checking; normal gene sequence in sequencing result and gene pool is compared, and observes with or without transgenation.

PCR reacts sequencing result and finds: FATP1c.68C>G, p.Pro23Arg all patients in family occur, in family, the normal person of all phenotypes is all without this sudden change (as shown in Figure 4); FLI1c.74C>T, p.Ala25Val also occur except occurring family troubles person in I 3 and IV 1; C.4819C>T, p.Arg1607Cys also occurs except occurring family troubles person CACNA1H in II 9.To sum up only have in the change point of FATP1 patient all in MRS family show as to be divided into disease from.We have detected 200 normal controls simultaneously, do not find this sudden change.

In addition, inventor has devised the primer for all exon regions of FATP1, performing PCR of going forward side by side detects, and to determine that FATP1 is with or without other mutational sites, primer is as following table:

The preparation of PCR reaction system and reaction conditions set the same.Other mutational sites are not found in this MRS family and normal control population.Contriver determines that MRS Disease-causing gene is FATP1 in the world first thus.

Embodiment 5: the test kit of the FATP1 gene of vitro detection MRS patient

In order to detect the pathogenic mutation of other this gene of MRS family, the primer sequence of all exons in this gene coding region and exon and intron intersection can be designed.In this embodiment, test kit primer and amplification order-checking condition are as embodiment 4.

1, test kit composition:

Primer: as described in Example 4;

Taq enzyme

Damping fluid

dNTP

Isolation and purification genetic material (DNA sample) from organism sample to be detected

2, using method:

(1) extracting genome DNA: use the Wizard GenomicDNA of Promega company of the U.S. to extract test kit and extract peripheral blood sample genomic dna.

(2) above-mentioned PCR primer, Taq enzyme, sample genomic dna, damping fluid, dNTP etc. is first adopted to carry out pcr amplification reaction;

(3) purifying is carried out to pcr amplification product;

(4) BigDye reaction is carried out to the PCR primer of purifying;

(5) to BiyDye reaction product purifying;

(6) to the order-checking of BiyDye reaction product, sequencing sequence is compared with normal sequence.

Claims (10)

1. a FATP1 gene for sudden change, is characterized in that: the FATP1 gene order of described sudden change has at least 1 non-silent mutation compared with SEQ ID NO:1, and the FATP1 gene of described sudden change causes the generation of Melkersson Rosenthal syndrome; Described non-silent mutation be selected from insertion, disappearance and displacement in one or more.

2. the FATP1 gene of sudden change as claimed in claim 1, is characterized in that: described non-silent mutation is that the C of the 68th of SEQ ID NO:1 sports G, and its sequence is as shown in SEQ ID NO:2.

3. a protein for the FATP1 genes encoding of sudden change as claimed in claim 2, is characterized in that: the aminoacid sequence of described protein is as shown in SEQ ID NO:3.

4. a carrier, is characterized in that: described carrier is containing, for example the FATP1 gene of the sudden change described in claim 1 or 2.

5. a genetically engineered host cell, is characterized in that: described host cell is the host cell of vector according to claim 4 or transduction.

6. the FATP1 gene of sudden change as claimed in claim 1 or 2 is as the drug target for the treatment of Melkersson Rosenthal syndrome or the application prepared in Melkersson Rosenthal syndrome kit for diagnosing diseases.

7. for diagnosing a test kit for Melkersson Rosenthal syndrome, it is characterized in that: described test kit comprises can the primer of specific amplification FATP1 gene, or can the probe of specific detection FATP1 transgenation.

8. test kit as claimed in claim 7, is characterized in that: the sequence that described primer or probe sequence are the 68th front and back 15 ~ 30bp length in the sequence shown in SEQ ID NO:1 or 2.

9. an antibody, is characterized in that: described antibody is combined with protein specific according to claim 3, and does not act on the protein of normal FATP1 coded by said gene.

10. a Melkersson Rosenthal syndrome therapeutical agent, described therapeutical agent comprises antibody according to claim 9.